Steroid hormone receptors are complex proteins which play a central role in steroid hormone action. Studies of these proteins in crude systems have suggested that all receptors have at least two functional domains: one for hormone binding, and one for interaction with DNA. The studies detailed in the present proposal are designed to purify receptors for progesterone and 1,25-dihydroxy vitamin D and to investigate the structure of these proteins and the regions of each involved in hormone and DNA binding. Receptor purification will include steroid affinity chromatography using a novel approach for elution of receptors from affinity resins: hormone displacement with mercurial reagents. This technique, which has been demonstrated in crude systems (Coty W. A. (1980) J. Biol. Chem. 255:8035-8037) has the potential for rapid and complete displacement of receptor from the resin under gentle conditions (low temperature and ionic strength); and, after addition of excess thiol agent, of recovering hormone-binding activity in high yield. Moreover, the mercurial elution technique has the unique potential for purifying apo-receptors and "unactivated" receptor forms, unlike other currently available purification techniques. The purified progesterone receptor proteins will then be used for affinity labelling studies using chemically- and photochemically-reactive steroid and nucleotide derivatives, in order to identify proteolytic fragments and amino acid residues involved in the hormone- and DNA-binding domains. Finally, these purified receptor proteins will be used to develop monoclonal antibodies, which can be used as further probes of the functional domains of the progesterone and 1,25-dihydroxy vitamin D receptors. In addition, these antibodies can be used in future studies of hormone action, such as immunocytochemical localization of receptor proteins. Completion of these aims will lead to a greater understanding of the mechanism of steroid hormone regulation of gene expression.